Substantial anti-tumor effects have been observed in patients with B-cell lymphoma treated with murine monoclonal antibodies directed at the idiotype of the cell surface immunoglobulin on malignant B-cells. Although this type of therapy represents an exciting approach to the treatment of B-cell malignancies, much needs to be learned regarding the reasons for the success or failure of such a therapy. Among the critical factors which require study are the characteristics of anti-idiotype antibodies which may play a role in the eventual anti-tumor response and the mechanisms whereby tumor cells escape control by these antibodies. We have focused on defining the role of such characteristics as antibody isotype, epitope specificity, affinity and antigenic modulation capacity using anti-idiotype antibodies in a murine model of B-cell lymphoma, 38C13. To date, the role of antibody isotype has been unambiguously defined in this model and a novel anti-idiotype effect of immunoselection for the outgrowth of idiotype variants which are no longer reactive with the therapeutic antibody has been discovered. Moreover, it has been determined that the immunophenotype of escaping tumors can serve as an additional marker of anti-tumor effects, that the isolated variants can be used to more precisely define antibody epitope specificity, and that escaping variant clones express a different light chain gene from the parental tumor. A large panel of new monoclonal anti-idiotype antibodies has recently been generated which contains antibodies with distinct differences in epitope specificity and binding affinity with respect to the parent tumor and its variants. We now propose to more precisely define our newly derived panel of antibodies with respect to affinity and epitope specificity. This will involve quantitation of binding constants by Scatchard analysis, binding competition assays, and Western blotting. The relative in vitro effects of these antibodies on proliferation of target tumor cells, cell surface idiotype antigen expression (antigenic modulation), antibody-dependent cellular cytotoxicity, and complement-dependent lysis of tumor cells will be analyzed. Selected antibodies of interest, because of in vitro findings, will be further assessed in vivo for their anti-tumor effects. These effects will be evaluated by analyzing the survival time of tumor-bearing animals and by immunophenotypic analysis by flow cytometry of escaping tumors. This latter assay will be used to determine the relative proportion of """"""""wild type"""""""" tumor cells to idiotype variant tumor cells in escaping tumor populations. Antibodies matched for isotype but differing in anti-proliferative effects, epitope specificity, affinity, and modulation capacity will be compared in these in vivo studies. Finally, the nature of tumor cells escaping antibody therapy will be further investigated on an immunophenotypic and molecular level. Studies will be directed at determining whether the process of idiotype variant generation is an ongoing precess, what its frequency is, and what molecular relationships exist between the variants selected out by various therapeutic antibodies. Protein studies of idiotype variant molecules by SDS-PAGE, DNA analysis by Southern blotting of immunoglobulin gene rearrangements, and direct nucleotide sequencing by the polymerase chain reaction technique will be performed on variant clones. The above studies will hopefully provide important and relevant information on the rational selection of antibodies for therapy of lymphoma as well as other malignancies and shed light on the heterogeneity and immunobiology of lymphoma.
